The human haploid genome is a deoxyribonucleic acid (DNA) sequence and consists of approximately three billion base pairs grouped into 23 chromosomes. However, the human genome is diploid and consists of approximately six billion base pairs grouped into 46 chromosomes. Hence, two copies of each genomic segment and two copies of each chromosome are represented in most of the human genome. The exception is the male human, which has only one copy each of chromosome X and chromosome Y. Nevertheless, variable copy numbers (i.e. not two copies) of genomic segments and chromosomes are observed in individual genomic DNA (gDNA) samples. Such copy number variable regions (CNVR) that are typically greater than one kilobase in length and generally occur at a minor frequency of equal to or greater than 1% in the population are termed “copy number variants” (CNVs; see, e.g., Feuk, et al. Nat. Rev. Genet. 7:85-97 (2006)). Copy number variation (CNV) and its mechanisms of formation, associations with phenotype, and methods of analysis have been extensively reviewed (Feuk, supra; Freeman, et al. Genome Res. 16:949-961 (2006); Sharp, et al. Annu. Rev. Genomics Hum. Genet. 7:407-442 (2006); Nature Genetics 39:S1-S54 (2007), entire issue). Currently, approximately 8600 CNVs have been identified and cover about 5-10% of the human genome (Redon, et al. Nature 444:444-454 (2006); Conrad, et al. Nature 464:704-712 (2010)). Continuing studies toward finer mapping of the CNV map and interrogating more diverse gDNA samples are tracked at the Database of Genomic Variants (http://projects.tcag.ca/variation/). Aberrations in the normal complement of 46 human chromosomes have been identified by cytogenetic analysis. Cytogenetics and its history, linking of chromosomal defects and disease, and methods of analysis have been extensively reviewed (Speicher, et al. Nat. Rev. Genet. 6:782-792 (2005); Trask, Nat. Rev. Genet. 3:769-778 (2002)). More recently, polymerase chain reaction (PCR)-based assays have been used to identify such aberrations.
There is a need in the art for methods that provide fast, accurate, easy-to-use and reliable karyotype information. Karyotype includes an analysis of chromosome number, type, shape, and banding. Currently available methods for determining karyotype and chromosome number lack accuracy due to the presence of CNVRs, are labor-intensive and do not provide for simultaneous interrogation of multiple chromosomes without requiring multiple reporting labels. Unless chromosomes are interrogated outside of CNVRs, an inaccurate karyotype or chromosome number determination may result. Accordingly, this disclosure provides such methods by using as target sequences only those targets known to be outside of CNVRs. In addition, the methods described herein provide for simultaneous interrogation of multiple chromosomes using a single, or multiple reporting labels. Using these methods, karyotypes may be rapidly and accurately determined. These and other advantages may be drawn from the description provided below.
For FIGS. 1-8, the genomic DNA (gDNA) samples on the x-axis from left-to-right are as follows:
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